Structural characterization of recombinant human interferon-gammas derived from two different mammalian cells

Area of Science:

• Biotechnology
• Protein Chemistry
• Cell Biology

Background:

• Recombinant human interferon-gamma (rHuIFN-gamma) is a crucial therapeutic protein.
• Mammalian cell lines are commonly used for producing rHuIFN-gamma.
• Understanding post-translational modifications is key to ensuring protein quality and function.

Purpose of the Study:

• To compare the structural properties of rHuIFN-gamma produced in two different mammalian cell lines: mouse C127 and Chinese hamster ovary (CHO) cells.
• To investigate the impact of host cell post-translational modifications on rHuIFN-gamma heterogeneity.

Main Methods:

• Microcarrier culture system for cell cultivation.
• Sequential chromatography for protein purification.
• Peptide mapping with V8 protease and Western blot analysis.
• Identification of glycosylated peptides.
• C-terminal and N-terminal amino acid sequence analysis.

Main Results:

• Mouse C127 cells produced a predominant ~25kDa rHuIFN-gamma component.
• CHO cells yielded both ~25kDa (di-glycosylated) and ~20kDa (mono-glycosylated) components.
• Both cell types produced rHuIFN-gamma variants with heterogeneous C-termini (lacking 2-16 amino acids).
• N-termini were blocked and appeared identical to natural HuIFN-gamma across both cell types.

Conclusions:

• Host cell type significantly influences post-translational modifications of rHuIFN-gamma.
• Glycosylation patterns and C-terminal processing differ between C127 and CHO cells.
• These variations contribute to the observed molecular heterogeneity of recombinant human interferon-gamma.